<p>Hirschsprung’s disease (HSCR) is a congenital disorder of the distal intestine characterized by aganglionosis of the enteric nervous system. While defective migration of neural crest-derived precursors is a well-established hallmark, how the intestinal microenvironment contributes to impaired neuronal support remains poorly understood. Here, we combined histochemical analyses of human HSCR colon with functional assays to investigate the role of muscularis macrophages (MMs) and a miR-93-5p–AHNAK pathway in regulating enteric neurons. Using immunohistochemistry and immunofluorescence on ganglionic and aganglionic segments, we mapped the density, spatial distribution, and phenotype of MMs, and quantified the expression of miR-93-5p and its predicted target AHNAK. In vitro, polarized M2-like macrophages were cocultured with enteric neuronal cell lines to assess neuronal migration, proliferation, and apoptosis. Gain-of-function and loss-of-function approaches for miR-93-5p, together with a dual-luciferase reporter assay, were used to validate AHNAK as a direct target. M2-like MMs (CD163⁺/CD206⁺) were abundant around myenteric ganglia in ganglionic colon but reduced in aganglionic segments, where AHNAK expression was increased. M2 macrophages enhanced neuronal migration and proliferation and protected against apoptosis, whereas disruption of the miR-93-5p–AHNAK axis impaired these neuro-supportive effects. Together, our data identify a muscularis macrophage–miR-93-5p–AHNAK axis that supports enteric neurons and demonstrates that loss of M2-like MMs and dysregulated miR-93-5p–AHNAK signaling compromise neuronal homeostasis in HSCR. These findings add a histochemical microenvironment perspective to HSCR pathogenesis and support a candidate neuroimmune pathway for restoring immune–neuronal balance, pending in vivo and clinical validation.</p>